The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H(+) and Na(+) as coupling ions. The sequence of the c subunit of V. cholerae F(0)F(1) ATPase suggested that this enzyme is H(+) specific, in contrast to the results of previous studies on the Na(+)-dependent ATP synthesis in closely related Vibrio spp. Measurements of the pH gradient and membrane potential in membrane vesicles isolated from wild-type and DeltaatpE mutant V. cholerae show that the F(1)F(0) ATPase of V. cholerae is an H(+), not Na(+), pump, confirming the bioinformatics assignments that were based on the Na(+)-binding model of S. Rahlfs and V. Müller (FEBS Lett. 404:269-271, 1999). Application of this model to the AtpE sequences from other bacteria and archaea indicates that Na(+)-specific F(1)F(0) ATPases are present in a number of important bacterial pathogens.